Hematopoietic growth factors exert their effects on normal and leukemic bone marrow progenitors by initially binding to specific cell-surface receptors. Cytosolic protein kinases are plausible links between receptor-ligand interactions and growth factor-specific cellular responses. The growth factors IL-3, GM-CSF and erythropoietin induce a rapid, dose-dependent phosphorylation of Raf-1, a 74kD cytosolic ser/thr protein kinase encoded by the protooncogene c-raf. Furthermore, in association with Raf-1 phosphorylation, IL-3, GM-CSF and erythropoietin mediate a marked increase in Raf-I kinase activity as measured in immune complex kinase assays in vitro. Additionally, a c-raf antisense oligonucleotide profoundly decreases intracellular Raf-1 levels and specifically inhibits growth factor-mediated proliferation. These data support a central and necessary role for Raf-1 in post-receptor hematopoietic growth factor signal transduction. Proposed plans are to: 1) Further define the mechanism(s) by which Raf-1 phosphorylation and activation occurs. The effects of hematopoietic growth factors, specific protein kinases, and oncogenes on the Raf-I phosphorylation state and protein kinase activity will be evaluated in primary leukemic cells and cell lines by Raf-1 immunoprecipitation and in vitro using purified Raf-1; 2) Determine the specific effects of c-raf antisense oligonucleotides on hematopoietic and leukemic cell growth; 3) Characterize the intracellular Raf-1 binding protein(s) including the potential Raf-1 substrate(s). The long-range goal is to partially sequence and clone the candidate Raf-1 substrate molecule(s). Ultimately, it is hoped that the understanding of the molecular components of hematopoietic cell growth will provide a rational basis for the design of effective and less toxic antileukemic therapy.